Binding-Induced Activation of DNA Alkylation by Duocarmycin SA: Insights from the Structure of an Indole Derivative−DNA Adduct

The mechanism for catalysis of DNA alkylation by the potent antitumor antibiotic duocarmycin SA (DSA) has been probed by determining the structure of a DNA adduct of the indole analogue (DSA-indole, DSI) lacking three methoxy functional groups. The three-dimensional structure of DSI covalently bound to A 19 in d-(G 1 AC TAATT GAC 11 )·d-(G 12 TC AATTA GTC 22 ) was determined by 1 H NMR spectroscopy using a total of 935 experimental distance and dihedral angle constraints. The representative ensemble of 20 conformers has no distance restraint violations greater than 0.03 Å, no torsional restraint violations greater than 0.7°, and a pairwise rmsd over all atoms in the binding site of 0.48 Å. Comparison of the structures of the DSA and DSI adducts reveals a structural basis for the critical role of one of the trimethoxy-indole functional groups in alkylation reactivity. A deeper penetration into the DNA minor groove in the vicinty of the indole subunit is observed for the DSI versus the DSA adduct, along with some variations in the width and depth of the minor groove throughout the binding site. The most significant difference between the DSI and DSA addducts is the 8° smaller twist of the two ligand subunits in DSI, which correlates with its ∼20-fold slower rate of DNA alkylation. This comparison of the structures of the DSI and DSA adducts to the same DNA duplex provides the most direct evidence to date in support of the proposal that the binding of the ligand in the DNA minor groove and consequent twisting of the two ligand subunts, disrupting vinylogous amide stabilization and thereby activating the conjugated cyclopropane electrophile, plays a central role in controlling DNA alkylation reactivity.